1 The long-term goal of this proposal is to understand how specific protein:protein interactions regulate the localization and function of protein kinase C (PKC) and protein kinase D (PKD). These two kinase families play pivotal roles in transducing the myriad of extracellular signals that promote phospholipid hydrolysis or, in the case of atypical PKC isozymes, 3'-phosphoinositide generation. We will focus on 1] the C-terminus of PKC as a paradigm for a molecular switch that controls kinase function through specific protein interactions, 2] identifying scaffolds for the atypical PKC isozymes and PKD isozymes, testing the hypothesis that these kinases have PDZ-binding motifs, and 3] uncovering mechanisms for lipid second messenger signaling in the matrix of mitochondria. Specifically, the following Aims are proposed: 1. The C-terminus of PKC as a molecular switch -The goal of this section is to understand how the interaction of specific binding partners with the C-terminal tail of PKC controls the lifecycle of this kinase. In the current funding period, and in collaboration with Pat Jennings (Project 4), we discovered that the prolyl isomerase Pin1 converts PKC into a species that can be degraded following activation. This Aim focuses on understanding the structural (with Project 4), biochemical, and cellular mechanisms underlying the molecular switch in the C-terminus of PKC. 2. Regulation of atypical PKC and PKD signaling by protein scaffolds - The goal of this section is to understand how protein interactions confer specificity in signaling by atypical PKC isozymes and PKD isozymes. We propose to identify PDZ domain scaffolds for these kinases and test how disruption of scaffolding interactions impact signaling. 3. Signaling at intracellular organelles: the mitochondria! matrix - The hypothesis driving this aim is that lipid second messenger signaling is a key component in mitochondria! function. Taking advantage of the novel genetically-encoded kinase activity reporters we developed in the current funding period, we aim to unambiguously demonstrate kinase activity in the matrix of mitochondria, an unexplored signaling terrain. We ask how disrupting kinase function at this location compromises mitochondria! function.